Published online 13 September 2013
Nucleic Acids Research, 2013, Vol. 41, No. 22 10323–10333
doi:10.1093/nar/gkt813
DNA polymerase d stalls on telomeric lagging
strand templates independently from
G-quadruplex formation
Justin D. Lormand1, Noah Buncher1, Connor T. Murphy1, Parminder Kaur2,
Marietta Y. Lee3, Peter Burgers4, Hong Wang2, Thomas A. Kunkel5 and
Patricia L. Opresko1,*
1
Received May 14, 2013; Revised August 16, 2013; Accepted August 19, 2013
ABSTRACT
Previous evidence indicates that telomeres
resemble common fragile sites and present a challenge for DNA replication. The precise impediments
to replication fork progression at telomeric TTAGGG
repeats are unknown, but are proposed to include
G-quadruplexes (G4) on the G-rich strand. Here we
examined DNA synthesis and progression by the
replicative DNA polymerase d/proliferating cell
nuclear antigen/replication factor C complex on
telomeric templates that mimic the leading C-rich
and lagging G-rich strands. Increased polymerase
stalling occurred on the G-rich template,
compared with the C-rich and nontelomeric templates. Suppression of G4 formation by substituting
Li+ for K+ as the cation, or by using templates with
7-deaza-G residues, did not alleviate Pol d pause
sites within the G residues. Furthermore, we
provide evidence that G4 folding is less stable on
single-stranded circular TTAGGG templates where
ends are constrained, compared with linear oligonucleotides. Artificially stabilizing G4 structures on
the circular templates with the G4 ligand BRACO-19
inhibited Pol d progression into the G-rich repeats.
Similar results were obtained for yeast and human
Pol d complexes. Our data indicate that G4 formation is not required for polymerase stalling on
telomeric lagging strands and suggest that an
alternative mechanism, in addition to stable G4s,
contributes to replication stalling at telomeres.
INTRODUCTION
Telomeres are nucleoprotein structures that prevent
degradation at chromosomes ends and chromosome endto-end fusions. Human telomeres consist of linear arrays
of 5–10 kb of TTAGGG repeats and end in a 30 singlestrand overhang. These repeats are necessary binding sites
for the shelterin protein complex, which isolates telomeres
from the DNA damage surveillance machinery (1). Loss of
telomeric DNA or shelterin proteins cause chromosome
ends to be recognized as DNA double-strand breaks,
which leads to growth arrest or chromosome alterations
(2). Telomeres shorten with each cell division owing to the
inability to completely replicate chromosome ends (3),
which is compensated for by telomerase. However, most
human somatic cells lack telomerase activity and the
majority of the telomere is maintained through semiconservative DNA replication during cell division (4).
Therefore, maintaining replication fork progression
through repetitive telomeric templates is critical for
preserving chromosome ends and overall genome stability.
Emerging data reveal that telomeres resemble common
fragile site sequences in the genome because they are prone
to alterations or breakage under replication stress conditions (5–7). The inhibition of replicative DNA polymerases or defects in replication stress checkpoints, generates
breaks at common fragile site sequences and causes
*To whom correspondence should be addressed. Tel: +1 412 624 8285; Fax: +1 412 624 9361; Email: [email protected]
ß The Author(s) 2013. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial
re-use, please contact [email protected]
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Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public
Health, 100 Technology Drive, Pittsburgh, PA 15219, USA, 2Department of Physics, North Carolina State
University, 2401 Stinson Drive, Raleigh, NC, 27695, USA, 3Department of Biochemistry and Molecular Biology,
New York Medical College, Valhalla, NY 10595, USA, 4Department of Biochemistry and Molecular Biophysics,
Washington University School of Medicine, St. Louis, MO 63110, USA and 5Laboratory of Molecular Genetics
and Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, DHHS, Research
Triangle Park, NC 27709, USA
10324 Nucleic Acids Research, 2013, Vol. 41, No. 22
Dr Walter Chazin. Recombinant protection of telomeres
1 (POT1) protein was purified using a baculovirus/insect
cell expression system and an AKTA Explorer FPLC (GE
Health Care, Piscataway, NJ, USA) as described previously (27).
DNA substrates
Oligonucleotides were purchased from Integrated DNA
Technologies and were purified using polyacrylamide gel
electrophoresis by the manufacturer. The oligonucleotide
with two 7-deazaguanine residues was purchased from
Midland Certified Reagents Company. The sequences of
the various primers are indicated in Supplementary
Table S1. Primers were 50 -end-labeled with [g-32P]ATP
(3000 Ci/mmol) (PerkinElmer Life Sciences) using
Optikinase (Affymetrix), according to the manufacturer’s
protocol. The telomeric sequences were inserted into
pGTK4 vector, which is a derivative of pGEM3zf()
(Promega Corporation) as described (28). Log-phase
cultures of plasmid-bearing E. coli strain DH5aIQ were
infected with R408 helper phage for 3 h for the generation
of ssDNA, which was purified as described (29). Primers
were annealed to ssDNA vectors in 0.4 saline-sodium
citrate, which equates to 60 mM NaCl, unless otherwise
indicated in the figure legend. Linear oligonucleotide
templates were prepared by annealing primers in either
100 mM KCl or 100 mM LiCl.
Atomic force microscopy analysis of G4 formation
Circular duplex DNA substrates containing a 157-nt
region of ssDNA were prepared as described previously
(30). The ssDNA region contained either (TTAGGG)10 or
CCTAA(CCCTAA)9C sequences flanked by 28 50 and 69
30 nucleotides. The linear (TTAGGG)10 DNA molecules
were constructed by annealing the Tel10 oligonucleotide
with the 6-nt running start (RS) primer (Supplementary
Table S1). DNA substrates were diluted to a final concentration of 1 mg/ml in a buffer containing 25 mM Hepes, pH
7.5, 25 mM Na-acetate and 10 mM MgCl2 and deposited
onto a freshly cleaved mica surface (SPI Supply). All
samples were washed with MilliQ water and dried under
a stream of nitrogen gas. Images were collected in tapping
mode using a MultiModeV microscope (Bruker
Corporation) with an E scanner or a MFP-3D-BIO
atomic force microscopy (AFM; Asylum Research).
PPP-NCL or PPP-FMR AFM probes (Nanosensors)
were used. Images were captured at a scan size of
1–4 mm2, a scan rate of 1–2 Hz and a resolution of
512 512 pixels.
DNA polymerase reactions
MATERIALS AND METHODS
Protein purification
Recombinant yeast Pol d was purified as described (24),
and recombinant yeast PCNA and RFC were purified
from Escherichia coli overproduction strains as described
(25). Recombinant 4 subunit, human Pol d and PCNA
were purified as previously described (26). Recombinant
human replication protein A was kindly provided by
Standard reactions for yeast Pol d contained 20 mM
Tris, pH 7.8, 0.2 mg/ml bovine serum albumin, 1 mM
dithiothreitol (DTT), 90 mM NaCl (unless otherwise
indicated),
10 mM
magnesium
acetate,
100 mM
deoxynucleotide (dNTP), 0.5 mM ATP and 6.6 nM
DNA substrate. Where indicated in the figure legend,
the DNA substrate was preincubated with 20 nM PCNA
and 13 nM RFC for 5 min at 30 C, and the reactions were
initiated by adding Pol d. Standard reactions for human
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telomere doublets at chromosome ends that resemble fragmented telomeres (5,8). Defects in telomere replication can
also yield chromatid ends that lack a telomere signal, presumably owing to telomere loss caused by failures in
telomere replication (9,10). Therefore, telomeres and
fragile site sequences share the common feature of being
challenging to replicate.
The precise obstacles to telomere replication are not
well defined. However, evidence from yeast and mammalian cells indicate that shelterin proteins suppress fork
stalling during telomere replication (5,8). Shelterin
proteins can recruit specialized DNA helicases including
Bloom Syndrome helicase (BLM), RTEL and Werner
syndrome helicase (WRN) to the telomeres, and these
helicases also suppress telomere fragility or stochastic
telomere loss, which suggests that secondary DNA structures impede replication (5,9,11–13). Tandem TTAGGG
single-stranded repeats can fold into G-quadruplex (G4)
DNA, which are four-stranded DNA structures that are
stabilized via Hoogsteen base pairing between guanines
(14). Because telomeres lack a known origin of replication,
the G-rich template is replicated by lagging strand DNA
synthesis and may fold into G4 DNA in the singlestranded DNA (ssDNA) gaps of Okazaki fragments.
WRN is required to prevent the loss of telomeres
replicated from the G-rich lagging strand in human cells
(9,15), and many helicases can unwind G4 structure
in vitro (16,17). Bimolecular G4 folds in the (CGG)n
sequence were shown to block DNA polymerase d (Pol
d) progression (18), which is the enzyme primarily responsible for replicating the lagging strand template (19).
Collectively, these data led to the model that replication
forks stall at telomeres owing to the propensity for G4
folding, and that specialized DNA helicases promote
telomere replication by unwinding G4 DNA (5,20–22).
However, other potential obstacles exist at telomeres
(23), and DNA synthesis by Pol d on lagging or leading
strand telomeric templates had not been examined.
Here we directly tested the model that Pol d stalls
during DNA synthesis on telomeric lagging strand DNA
templates owing to G4 folding. Consistent with previous
predictions, we observed that Pol d pauses at the G runs
within TTAGGG repeats, even in the presence of the
proliferating cell nuclear antigen (PCNA) processivity
clamp and replication factor C (RFC) clamp loader.
Surprisingly, stalling is still observed even under conditions that suppress G4 folding. Our data indicate that
besides G4 formation, other mechanisms, such as
specific sequence context effects, may contribute to
telomere fragility during DNA replication.
Nucleic Acids Research, 2013, Vol. 41, No. 22 10325
RESULTS
Pol d DNA synthesis on telomeric templates
We first examined telomeric lagging strand DNA synthesis
by Pol d from Saccharomyces cerevisiae. Although the
telomeric sequence in budding yeast differs from human,
the telomere lagging strand template is G-rich (TG1-3)
and forms G4 DNA in vivo (31). Furthermore, replication
forks stall at human telomeric repeats in yeast whether the
TTAGGG repeats are engineered into a plasmid or at
chromosome ends (32,33). Circular ssDNA templates
were prepared from phagemids containing 10 telomeric
repeats inserted in either orientation to generate lagging
(G-rich) or leading (C-rich) strand templates (Figure 1A).
We previously used these vectors to demonstrate that
human telomeric sequence is replicated accurately in
normal human cells (28). Oligonucleotide primers
allowed for a 3-nt running start (RS) (TCT sequence)
before the telomeric sequence. Potential structures that
can form within the TTAGGG repeats include G4 DNA
and intermediate hairpin or triplex folds (34,35) (Figure
1B). A 1:1 ratio of enzyme to template yielded sufficient
progression through the telomeric repeats to detect potential stalling. The time course confirmed the expected
multiple hit conditions, whereby the polymerase can
rebind and extend previously terminated products during
the 30-min reaction (Figure 2) (36). Thus, the bands representing accumulated products reveal prominent sites of
polymerase stalling. Pol d showed a distinct pausing
pattern at each G run on the lagging template (pausing
at the third G in each repeat Figure 2A and B), but was
more processive on the leading template. The strongest
pause site on the leading C-rich strand occurred after
the telomeric sequences. The Pol d 30 to 50 exonuclease
generated some primer degradation products on the Grich telomeric template; an average of 3.6, 2.5 and 1.7%
primer degradation after 5, 15 and 30 min, respectively.
Despite the strong pause site at the third and fourth
template positions on the nontelomeric template, no
primer degradation was detected (Figure 2).
Next we asked whether the replication processivity
clamp PCNA would prevent polymerase stalling. RFC
protein loads PCNA onto the circular templates. As predicted, PCNA increased the product lengths on all templates (Figure 2). PCNA reduced, but did not eliminate,
products of Pol d pausing on the G-rich template. PCNA
also partly suppressed Pol d primer degradation on the
G-rich template, with an average of 3.0, 0.63 and 0.15%
primer degradation after 5, 15 and 30 min (Figure 2). The
data show DNA synthesis on the G-rich template was a
challenge for the PCNA/Pol d complex.
Mechanism for Pol d stalling at TTAGGG repeats
To test whether an immediate barrier was responsible for
stalling and primer degradation on the G-rich template,
we used a primer that allowed for a 6-nt RS before the
telomeric sequence (Figure 1, gray primer). When polymerase progression is inhibited, the 30 primer terminus
shuttles from the polymerase active site to the exonuclease
active site, resulting in primer degradation (37). Moving
the potential barrier further downstream from the start
site completely alleviated Pol d primer degradation,
A
B
Figure 1. Templates for DNA synthesis. (A) The control, telomeric leading strand or telomeric lagging strand templates were generated by annealing
a primer to circular ssDNA. Primers created either a 3- (black line) or 6- (gray line) nt RS before telomeric repeat residues (underlined nucleotides).
Sequences of the first 66 template nucleotides are shown 50 to 30 . (B) Schematic of potential secondary structures in TTAGGG repeats. Hairpin and
triplex structures are proposed intermediates in G4 folding. Monovalent ions K+ or Na+ (gray balls) stabilize the G4.
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Pol d contained 40 mM Tris, pH 7.8, 0.2 mg/ml bovine
serum albumin, 1 mM DTT, 5 mM NaCl (unless otherwise
indicated), 10 mM MgCl2, 0.5 mM ATP, 250 mM dNTP
and 6.6 nM DNA substrate. Where indicated in the
figure legend, the DNA substrate was preincubated with
170 nM human PCNA and 13 nM RFC for 5 min at 37 C,
and the reactions were then initiated by adding Pol d. At
the specified time points, 8 ml aliquots were removed from
the reactions and terminated using 95% formamide and
5 mM EDTA. Reaction products were resolved on a 10%
denaturing polyacrylamide gel and visualized with a
Typhoon 9400 phosphoimager. Products and unused
primer were quantitated by ImageQuant software and
were corrected for background in the no enzyme control
reactions. The percent of primer degradation was
calculated as the amount of radioactivity in bands for
shortened primers divided by total radioactivity in the
lane. To analyze the extent of DNA synthesis, the
amount of radioactivity in bands for products terminated
before the telomeric sequence (RS) or for products
terminated within and beyond (50 ) the telomeric sequence,
was divided by the total amount of radioactivity in the lane.
10326 Nucleic Acids Research, 2013, Vol. 41, No. 22
A
Figure 2. Pol d stalls at TTAGGG repeats on lagging strand templates.
Reactions contained 6.6 nM circular ssDNA primed with a 32P endlabeled 40-mer to allow a 3-nt RS before telomeric sequence (see
Figure 1). Reactions were initiated by adding 6.6 nM Pol d and were
incubated under standard conditions and 90 mM NaCl at 30 C.
Aliquots were terminated at 5, 15 and 30 min and run on 10% polyacrylamide denaturing gels. (A) Gel images for reactions containing Pol
d alone or for templates preincubated with 20 nM PCNA and 13 nM
RFC. Thick black lines mark locations of the telomeric repeats, and Gs
mark the third guanine of each repeat. RS indicates the 3-nt RS, and P
marks the primer. (B) Prominent stall sites occurred at the third Gs in
the TTAGGG repeats on the template; marked with an arrow.
although stalling at G-runs remained (Supplementary
Figure S1).
To determine whether the apparent ‘barrier’ on the
G-rich template is G4 DNA, we repeated the reactions
under conditions that suppress or promote G4 formation.
Monovalent cations Na+ or K+ stabilize a G4 fold by
coordinating in the central cavity formed by the guanine
tetrads (Figure 1B); however, Li+ ions are too small for
efficient coordination and cannot stabilize the G4 (38).
Primers were annealed to templates in either 100 mM
KCl or LiCl, and 90 mM NaCl was substituted with
either 100 mM KCl or LiCl in the polymerase reactions.
Remarkably similar patterns of Pol d products were
observed in both ionic conditions. Surprisingly, substitution with LiCl did not alleviate Pol d stalling at G-runs or
Pol d inhibition is greater on linear compared with
circular (TTAGGG) templates in K+ buffer
The result that Pol d progression is not significantly
blocked on the (TTAGGG)4 templates is surprising
based on the wealth of structural data that shows
(TTAGGG)4 ssDNA forms stable G4 structures
(14,41,45). However, these studies used linear ssDNA.
Therefore, we tested Pol d DNA synthesis on linear templates containing either 10 or 4 telomeric repeats. PCNA
was omitted because it slides off linear templates; therefore, we tested various Pol d concentrations reacted for
30 min. Substrates were annealed in either 100 mM KCl
or LiCl, and included a 6-nt RS. The K+ buffer inhibited
Pol d progression on both the (TTAGGG)10 and
(TTAGGG)4 templates, compared with Li+ (Figure 4,
panels 1–2). K+ increased the percent of products terminated within the RS region by 2-fold on the (TTAGGG)10
templates, and by 7-fold on the (TTAGGG)4 templates at
the highest Pol d concentrations. K+ also increased the
percent of degraded primers for both templates [0.53%
(Li+) versus 3.6% (K+) on the (TTAGGG)4 templates at
6.6 nM Pol d]. We replaced the middle G with 7-deazaG in
the first and third repeats to prevent Hoogsteen bonding
and G4 folding (34). This eliminated the K+-dependent
Pol d inhibition; however, stalling at the G repeats
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B
the primer degradation (Figure 3A). The average % primer
degradation was 1.9 and 2.7% for K+ and Li+, respectively,
at 5 min. While reactions under optimal G4 folding conditions (KCl) did not enhance Pol d stalling, it caused a near
2-fold increase in products terminated within the 3-nt RS
before the repeats, compared with LiCl (Figure 3B). This
suggests that KCl was promoting some G4 folding that
partly impeded polymerase progression.
Previous reports showed that K+ induces greater G4
stability and DNA polymerase arrest at G4-forming sequences, compared with Na+ (39,40). Thus, the result that
100 mM KCl does not significantly block Pol d progression on the (TTAGGG)10 templates was surprising. Some
evidence suggests that the number of telomeric repeats
affects G4 stability, and that molecules with greater
repeat numbers are less thermally stable than molecules
with four repeats (41). Therefore, we predicted that
reducing the repeat number to four would enhance the
inhibition of Pol d progression. Unexpectedly, Pol d synthesis on the (TTAGGG)4 template was more similar
under the different ionic conditions (K+ versus Li+),
compared with the (TTAGGG)10 template (Figure 3C
and Supplementary Figure S2A). Furthermore, the percent
of products terminated within the RS was lower for the
(TTAGGG)4 template, compared with the (TTAGGG)10
template (Figure 3B and C). Increasing the RS to 6 nt did
not alter the results (Supplementary Figure S2B).
Collectively, our data indicate that G4 formation is not
required for Pol d stalling on telomeric lagging strand
templates. Consistent with this, neither ssDNA binding
protein replication protein A nor telomeric ssDNA
binding protein POT1 prevented Pol d stalling on the
TTAGGG templates (data not shown), despite the
ability of these proteins to disrupt G4 folds (42–44).
Nucleic Acids Research, 2013, Vol. 41, No. 22 10327
A
B
C
remained (Figure 4, panel 3). Nontelomeric control templates that lacked G4 folding ability confirmed that the
different ionic conditions do not alter Pol d catalysis
(Figure 4, panel 4). In summary, increased Pol d inhibition
on the linear templates with four repeats, compared with
10, is consistent with increased G4 folding stability.
The greater K+-dependent inhibition of Pol d DNA synthesis on linear compared with circular templates suggests
differences in G4 formation. To test whether nucleotides
flanking the TTAGGG repeats in the circles could pair
with the repeats and prevent G4 formation, we used
Mfold to identify potential secondary structures in the
polymerase reaction conditions (46). Two thermodynamically favored structures were predicted. One was an interrupted 7-bp hairpin immediately following the repeats,
and the other was an interrupted 7-bp hairpin involving the
last two repeats of either the (TTAGGG)4 or (TTAGGG)10
construct (Supplementary Figure S3). The latter hairpin
would prevent G4 folding only in the (TTAGGG)4 circle.
To eliminate hairpin formation, we annealed a 39-mer
oligonucleotide containing a blocked 30 terminus to the
50 flank of the repeats. No synthesis was observed past
the resulting 39-bp duplex (Supplementary Figure S4).
Using a 6-nt RS primer we observed only a 2-fold difference in products terminated within the RS region for reactions in K+, compared with Li+, and no difference after
30 min (Supplementary Figure S4D). Under the identical
conditions in K+, there was significantly more synthesis
past the repeats on the circular gaps (Supplementary
Figure S4A, lane 4), compared with the linear templates
(Figure 4, panel 2, lane 4) (81 ± 4.1% versus 42 ± 4.0%).
In contrast, synthesis past the repeats was similar in Li+
(81.2 ± 1.4% on circular gaps versus 82 ± 3.6 on linear
templates) (Figure 4 and Supplementary Figure S4).
Thus, interference by flanking nucleotides does not
explain why G4 formation appears to present a stronger
barrier to polymerase progression on telomeric linear templates compared with circular templates.
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Figure 3. Preventing G4 folding on telomeric lagging strand templates does not suppress Pol d stalling. Reactions contained 6.6 nM of circular
ssDNA templates containing 10 (A–B) or 4 (C and Supplementary Figure S2) TTAGGG repeats, primed with a 40-mer for a 3-nt RS. Primers were
annealed in either 100 mM KCl or 100 mM LiCl. Reactions were initiated by adding 6.6 nM Pol d alone or with 20 nM PCNA and 13 nM RFC, and
were conducted under standard conditions except NaCl was substituted with 100 mM KCl or 100 mM LiCl. Aliquots were terminated at 5, 15 and
30 min and run on denaturing gels. Thick lines mark the telomeric repeats. RS indicates the running start, P marks the primer and Gs indicate the
third guanine of each repeat. (B–C) Quantitation of Pol d/PCNA/RFC reactions on (TTAGGG)10 (B) or (TTAGGG)4 (C) templates. The percent of
primers extended past the RS were calculated as a function of total DNA. The percent of products terminated within the RS nucleotides were
calculated as a function of total DNA. K+, black line and squares; Li+, gray line and circles. Mean and standard deviations from two to three
independent experiments.
10328 Nucleic Acids Research, 2013, Vol. 41, No. 22
A
(TTAGGG)10
K+
(TTTAGGG)nTCTCGC
Li+
running start (RS)
7-deazaG (*)
(TTAGGG)4
K+
G
K+
Li+
non-telomeric
K+
Li+
G
G
G
G
G
*
G
G
G
G
G
*
Li+
G
RS
P
% products in RS
Pol
B
0
0
0
0
80
80
K+
60
K+
20
0
2
Pol
4
nM
0
0
K+
Li+
2
Pol
4
6
40
40
20
20
0
0
0
2
Pol
nM
4
nM
K+
Li+
60
Li+
20
6
0
80
60
40
Li+
0
80
60
40
0
0
6
0
0
2
Pol
4
6
nM
Figure 4. Pol d exhibits increased blockage on linear templates with 4 TTAGGG repeats compared with 10 in the presence of KCl. (A) Reactions
contained 6.6 nM linear ssDNA with 10 or 4 (TTAGGG) repeats, four repeats with two 7-deazagaunine substitutions (indicated by asterisk), or a
nontelomeric sequence (Supplementary Table S1). Templates were annealed with a primer in either 100 mM KCl or LiCl, for a 6-nt RS (see
schematic). Reactions were initiated with 0.41, 1.6 or 6.6 nM Pol d and were incubated under standard conditions except NaCl was replaced with
100 mM KCl or LiCl. Reactions were terminated at 30 min and run on denaturing gels. Thick lines mark repeats, RS marks the first 6 nt, P indicates
primers and Gs mark the third guanine of each repeat. (B) The percent of products terminated within the RS was calculated as a function of total
DNA. K+, black line and squares; Li+, gray line and circles. Mean and standard deviations from two to three independent experiments.
A (TTTAGGG)10
B CCTA(CCCTAA)9C
2 nm
2 nm
1 µm
1 µm
1 µm
2 nm
GAP
1 nm
0 nm
Figure 5. A greater fraction of linear templates exhibit stable G4 structures compared with circular templates. Representative AFM images of
circular gapped DNA substrates with (TTAGGG)10 ssDNA (A) or CCTAA(CCCTAA)9C ssDNA (B) regions. The image is 1 1 mm at 2 nm Z-scale.
The white arrow points to a characteristic G4 structure (height = 1.2 nm) on a circular (TTAGGG)10 gapped substrate.
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direction of synthesis
G
G
G
G
G
G
Nucleic Acids Research, 2013, Vol. 41, No. 22 10329
Pol d stalling at G-rich repeats is conserved
Next we asked whether human Pol d (hPol d) exhibits
similar stalling on the TTAGGG templates, compared
with yeast. We used hPol d and human PCNA amounts
that yielded similar levels of total primer extension on the
(TTAGGG)10 circles compared with yeast after 30 min
(95 ± 1.1% for yeast and 94 ± 1.3% for human).
Similar to yeast, hPol d also showed stalling at the third
G of each repeat and primer degradation on the lagging
strand TTAGGG template (Figure 6). While both
enzymes showed improved processivity on the C-rich
leading strand, compared with the lagging strand, the
human enzyme yielded more prominent stall products on
the leading strand corresponding to the third C of each
repeat (Figure 6 compared with Figure 2). The reactions
were conducted under the established optimal conditions
for hPol d of 5 mM NaCl; however, low monovalent ion
concentrations do not support stable G4 folding (29,50).
C-rich
- - - +++
- - -+++
C
C
C
C
C
G
G
C
G
C
G
C
G
C
G
C
G
RS
P
P
Time 0
Repeats
Direction of DNA synthesis
hPCNA
/RFC
G-rich
0
Figure 6. Pol d stalling within G-rich telomeric repeats is conserved.
Reactions contained 6.6 nM ssDNA primed with a 40-mer for a 3-nt
RS (see Figure 1). Reactions were initiated by adding 10 nM human Pol
d and were incubated under standard conditions with 5 mM NaCl at
37 C. Aliquots were terminated at 5, 15 and 30 min and run on
denaturing gels. Gel images are shown for reactions containing Pol d
alone or for templates preincubated with 170 nM human PCNA and
13 nM RFC as indicated. Thick lines indicate telomeric repeats, P
marks the primer, RS indicates the running start and Gs or Cs
indicate the third guanine or cytosine, respectively, in each repeat.
We observed that human Pol d showed greatly reduced
activity under high salt, precluding comparisons in
100 mM K+ versus Li+ reaction conditions (data not
shown).
To modulate G4 folding we added BRACO-19, a well
established G4 stabilizing ligand with high affinity for
telomeric G4 DNA (51). Reactions were conducted with
30 mM KCl or LiCl for 30 min with increasing BRACO19 concentrations. Reactions in K+ showed a slight
increase in products terminated within the RS and additional pause sites at TA residues, compared with reactions
in Li+ (Figure 7 compare lanes 2 and 9). The K+-dependent alterations were greatly enhanced by BRACO-19,
which induced a complete shift in pausing at Gs to
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Another possibility is that the frequency or stability of
G4 formation on linear molecules is greater than on
circular molecules. To test this we measured the fraction
of substrates showing G4 structures by AFM imaging.
Different DNA structures, including ssDNA, duplex
DNA and G4 folds, can be delineated on AFM images
by comparing molecular heights (44,47,48). We established previously that G4 structures in AFM images of
oligonucleotides containing TTAGGG repeats exhibit a
mean peak height at 0.9 (±0.2) nm (44). To examine the
frequency of forming stable higher order DNA structures
on circular templates, we prepared duplex plasmids
containing an ssDNA gap of 157 nt [(TTAGGG)10
sequence flanked by 28 and 69 nt of nontelomeric
sequence that cannot form G4]. Imaging of gapped
plasmids was required because ssDNA circular plasmids
appear condensed, perhaps owing to interactions between
complementary sequences (49). A minor population (15%,
N = 34) of the circular (TTAGGG)10 gapped molecules
displayed a region with peak heights >0.5 nm (average
1.1 ± 0.2 nm), consistent with the formation of G4 and
intermediate structures (Figure 5A) (44). When the
G-rich repeats were replaced with (CCCATT) repeats,
the gapped plasmids did not display any regions with
heights >0.5 nm (N = 20) (Figure 5B). AFM images of
linear (TTAGGG)10 molecules under the identical conditions revealed particles with mean peak heights of
0.79 ± 0.36 nm, consistent with secondary structures and
G4 folding (Supplementary Figure S6A and C) (44).
Substituting Li+ for Na+ yielded molecules with heights
at 0.24 ± 0.057 nm, which are less than double-stranded
DNA (0.37 ± 0.079 nm) and are consistent with ssDNA
(Supplementary Figure S5B and C). In contrast to circular
(TTAGGG)10 molecules, most of the linear (TTAGGG)10
molecules formed secondary structures consistent with G4
or intermediate structures (74%), indicated by heights
ranging from 0.5 to 1.63 nm (Supplementary Figure
S5C). Thus, a greater fraction of linear molecules with
TTAGGG repeats exhibit G4 structures, compared with
circular molecules, which corresponds with the increased
Pol d inhibition on linear templates.
10330 Nucleic Acids Research, 2013, Vol. 41, No. 22
A
B
C
Figure 7. G4 stabilizer BRACO-19 increases Pol d stalling and blockage on TTAGGG templates in the presence of K+. (A) The (TTAGGG)10
circular ssDNA substrate was annealed in either 100 mM KCl or LiCl with a primer for a 3-nt RS. Polymerase reactions contained 6.6 nM templates
preincubated with 0, 0.16, 0.32, 0.80, 4.0 or 8.0 mM BRACO-19, 170 nM hPCNA and 13 nM RFC. Reactions were initiated by adding 10 nM hPol d,
and were conducted in standard conditions except NaCl was substituted with either 30 mM KCl or LiCl. Aliquots were terminated at 5, 15 and
30 min and run on denaturing gels. Thick lines indicate telomeric repeats, P marks the primer, RS indicates the running start and Gs indicate the
third guanine in each repeat. Asterisk indicates shift to pause sites at TA sites. (B) The percent of primers extended past the RS. (C) The percent of
products terminated within the three RS. (D) The percent of unextended primers. K+, black line and squares; Li+, gray line and circles. Data are
mean and standard deviations from two to three independent experiments.
pausing at TA sites, and caused a 5-fold increase in
products terminated within the RS at 8 mM BRACO-19.
BRACO-19 did not induce these changes in reactions containing Li+; however, the highest ligand concentrations
caused some nonspecific inhibition of total DNA synthesis. Similar results were achieved with yeast Pol d
(Supplementary Figure S6). The results indicate that
both G4 folding and G4-independent mechanisms can
induce hPol d stalling on telomeric lagging strand
templates.
DISCUSSION
A great deal of evidence from mammalian cells and yeast
indicate that telomeres are difficult regions to replicate
(23). The G-rich lagging strand is preferentially lost in
the absence of some telomeric and DNA repair proteins
(9,10). The ability of (TTAGGG)4 sequences to form secondary G4 structures, and the result that G4 folds arrest
DNA polymerases in vitro, led to the model that
quadruplex formation impedes telomere replication
(5,9,20). Here we examined for the first time DNA synthesis on telomeric lagging strand templates by the lagging
strand replicative polymerase in vitro. We observed that
Pol d can proceed through the telomeric TTAGGG
repeats, but significant stalling occurs at G-runs within
the repeats even in the presence of PCNA processivity
factor. Stalling persisted after suppressing G4 formation
by altering monovalent ions or by manipulating the
template sequence. Our data suggest that telomere fragility and replication fork stalling in vivo (5) is due to pausing
of the lagging strand replicative DNA polymerase at
TTAGGG repeats. However, we provide strong evidence
that G4 folds are not required for the apparent stalling,
which raises the possibility that G4s are not the main
source of telomere replication stalling in vivo.
Given the wealth of structural data that shows TTAGGG
sequences can spontaneously fold into stable G4s (14,45),
the result that Pol d DNA synthesis is not significantly blocked at the TTAGGG repeats is surprising.
Biomolecular G4 folds of the sequence (CGGG)7 present
a strong block to yeast Pol d in 20 mM KCl (18).
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D
Nucleic Acids Research, 2013, Vol. 41, No. 22 10331
template may form the basis for the sequence context
effects that we observed. Both yeast and human Pol d
exhibit increased slippage errors in (GT)10 compared
with (TC)11 microsatellite sequences (57,58). Human Pol
d-PCNA does not stall at each G on a (GT)9 template, but
strongly pauses toward the end of the repeats for
unknown reasons (58). Together with our results, this
suggests that the topology of some G-rich tracts may be
problematic for Pol d.
Potential barriers in G-rich sequences include intermediates in G4 folding (Figure 1) that do not require
K+ or Na+. Hairpin and triplex structures can form in
TTAGGG repeats through Hoogsteen base pairing
between G residues (34,35), and both structures are
proposed to impede DNA synthesis in other contexts
such as genes responsible for triplet repeat diseases (59).
However, stalling at Gs occurred even on the (TTAGGG)4
templates containing two 7-deazaguanine substitutions
that prevent Hoogsteen base pairing (Figure 4, panel 3).
Single molecule studies revealed that unfolded (TTAGGG)4
molecules are significantly more compact than a polyT
construct, possibly owing to increased stacking between
purine bases or transient hydrogen bonding (35). We
propose that these transient attractive forces lead to
stalling at G residues on the TTAGGG template, and
that stabilized G4 folds lead to polymerase arrest or
stalling at TA residues (Figure 7 and Supplementary
Figure S6). This is because G4 stabilization with the
BRACO-19 ligand shifts the pause sites from the G runs
to the TA residues in the repeats (Figure 7). Structural
data indicates that BRACO-19 molecules interact
directly with the TA residues when bound to a telomeric
G4 (51). Our studies with BRACO-19 reveal both
stabilized G4-dependent and independent Pol d stalling
mechanisms on TTAGGG templates.
Our result that yeast Pol d stalls on telomeric templates
is consistent with reports that human telomeric sequences
in a plasmid or at chromosome ends induce replication
fork stalling in S. cerevisiae (32,33). Interestingly, these
studies found that RecQ helicase Sgs1 and Pif1 helicase
do not prevent replication stalling through human telomeric repeats, although these enzymes unwind G4 DNA
in vitro and influence replication and transcription in other
G4-forming sequence (17,60). Furthermore, mechanical
telomeric G4 unfolding studies indicate that only a few
base pairs need to be disrupted to destabilize the entire
G4 (35). Studies with an antibody that binds G4 structures
revealed that the majority of antibody staining occurred
outside the telomeres, suggesting that other G-rich
sequences in the genome form more stable G4s
compared with telomeric sequences (61). These findings
further support our conclusion that G4 formation may
not be the primary impediment to telomeric lagging
strand DNA synthesis, and suggest alternative mechanisms by which specialized helicases may promote telomeric
DNA replication in human.
In summary, cellular studies have established telomeric
repeats as difficult sequences to replicate in yeast and
mammalian cells (23), and our biochemical studies
indicate that the lagging strand replicative polymerase
stalls on TTAGGG templates. However, our data show
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In addition, previous studies showed that numerous DNA
polymerases arrest on G-rich templates owing to G4 formation in the presence of K+ or Na+, but not Li+ (40). Yet,
our results for both yeast and human Pol d differed. We
observed similar extension past the repeats in the presence
of K+ compared with Li+ on the (TTAGGG)10 circular
templates (Figures 3 and 7). One likely explanation is
that human TTAGGG repeats form less stable G4 folds
compared with the G4-forming sequences tested in
previous polymerase assays, which primarily used templates with a greater number of Gs in the repeats (39).
For example, TTGGGG repeats form more stable G4
folds compared with TTAGGG owing to an additional
tetrad of paired G residues (52). Another likely reason is
that increasing the number of TTAGGG repeats decreases
G4 stability (41), and increases the diversity of G4 conformations and the dynamics of folding and unfolding
(H. Hwang, J. Lormand, P. Opresko, and S. Myong,
submitted for publication). Consistent with this, we
observed greater Pol d inhibition on the linear templates
containing four repeats, compared with 10 repeats
(Figure 4). Notably, the nuclear magnetic resonance and
crystal structure data were collected from linear molecules
with four repeats (45,53). Thus, we expect that G4 folding
in the (TTAGGG)10 template is highly dynamic and that
mobile G4s may allow a polymerase to pass.
The context of TTAGGG repeats influences G4 folding,
and thus, also impacts the ability of Pol d to proceed
through the telomeric sequence. We observed that G4
structures are less stable, or fold less frequently, in
closed circles compared with linear substrates (Figure 5).
This likely explains why preventing G4 folding with Li+
minimally affected Pol d progression on the circular templates, but greatly inhibited progression on linear templates (Figures 3 and 4, Supplementary Figure S4).
Spatial constraints that exist in the closed circles, but
not in the open linear substrates, may influence G4
folding. The ends or borders of the TTAGGG region
are constrained within the circles, and how this may influence G4 folding and stability will require future biophysical and structural studies. While negative supercoiling in
duplex plasmids was found to generate regions of local
unwinding that allow G4 folding, the frequency of G4
folding compared with linear DNA had not been
compared (54). The majority of previous work examined
G4 stability within linear molecules (14,41). Our data
suggest that the spatial constraints that exist within replication bubbles on a chromosome may destabilize G4
folding, and lessen the impact of G4 folding on polymerase progression during telomeric DNA replication.
Because polymerase stalling occurs in the absence of G4
folding, other barriers must impede telomeric lagging
strand DNA synthesis. Sequence context effects on
DNA synthesis have been demonstrated for various
DNA polymerases; however, the molecular mechanisms
are not well understood (55). Evidence suggests Pol d
makes extensive contacts with the template ahead of the
active site. Structures of yeast Pol d and related
bateriophage polymerase RB69 reveal a channel in the
N-terminal domain that is proposed to bind 10–20 nt of
the unpaired template strand (56). These contacts with the
10332 Nucleic Acids Research, 2013, Vol. 41, No. 22
that G4 folding is not required for polymerase stalling
during telomeric DNA synthesis, and show that
introducing spatial constraints or increasing the number
of telomeric repeats reduces G4 stability. This raises the
possibility that the specialized DNA helicases WRN,
BLM and RTEL may facilitate telomere replication by
mechanisms other than unwinding G4 (9,13,22,62).
A better understanding of telomeric DNA replication is
required to elucidate mechanisms that lead to telomere
disruption and loss.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
We are grateful to Mercedes Arana and Gregory Sowd
for technical support, and to Sua Myong and Helen
Hwang for helpful discussion. We thank Walter Chazin
(Vanderbilt University) for generously providing recombinant human replication protein A (R01 GM65484).
FUNDING
NIH [R01ES0515052 to P.L.O., R01GM032431 to P.M.B.,
R00ES016758 to H.W., R01GM031973 to M.Y.L.] (in
part); Division of Intramural Research of the National
Institutes of Health, NIEHS [Z01 ES065070 to T.A.K.].
Funding for open access charge: Scaife Foundation
through the Center for Nucleic Acids Science and
Technology at Carnegie Mellon University (to P.L.O.).
Conflict of interest statement. None declared.
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